Soybean cultivar 03330181

ABSTRACT

A soybean cultivar designated 03330181 is disclosed. The invention relates to the seeds of soybean cultivar 03330181, to the plants of soybean cultivar 03330181, to the plant parts of soybean cultivar 03330181, and to methods for producing progeny of soybean cultivar 03330181. The invention also relates to methods for producing a soybean plant containing in its genetic material one or more transgenes and to the transgenic soybean plants and plant parts produced by those methods. The invention also relates to soybean cultivars or breeding cultivars, and plant parts derived from soybean cultivar 03330181. The invention also relates to methods for producing other soybean cultivars, lines, or plant parts derived from soybean cultivar 03330181, and to the soybean plants, varieties, and their parts derived from use of those methods. The invention further relates to hybrid soybean seeds, plants, and plant parts produced by crossing cultivar 03330181 with another soybean cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive soybean cultivar,designated 03330181. All publications cited in this application areherein incorporated by reference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single cultivar animproved combination of desirable traits from the parental germplasm.These important traits may include, but are not limited to, higher seedyield, resistance to diseases and insects, better stems and roots,tolerance to drought and heat, altered fatty acid profile, abioticstress tolerance, improvements in compositional traits, and betteragronomic quality.

These processes, which lead to the final step of marketing anddistribution, can take from six to twelve years from the time the firstcross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

Soybean (Glycine max), is an important and valuable field crop. Thus, acontinuing goal of soybean plant breeding is to develop stable, highyielding soybean cultivars that are agronomically sound. The reasons forthis goal are to maximize the amount of grain produced on the land usedand to supply food for both animals and humans. To accomplish this goal,the soybean breeder must select and develop soybean plants that have thetraits that result in superior varieties.

The soybean is the world's leading source of vegetable oil and proteinmeal. The oil extracted from soybeans is used for cooking oil,margarine, and salad dressings. Soybean oil is composed of saturated,monounsaturated, and polyunsaturated fatty acids. It has a typicalcomposition of 11% palmitic, 4% stearic, 25% oleic, 50% linoleic, and 9%linolenic fatty acid content (“Economic Implications of Modified SoybeanTraits Summary Report,” Iowa Soybean Promotion Board and AmericanSoybean Association Special Report 92S (May 1990)). Changes in fattyacid composition for improved oxidative stability and nutrition areconstantly sought after. Industrial uses of soybean oil, which issubjected to further processing, include ingredients for paints,plastics, fibers, detergents, cosmetics, lubricants, and biodiesel fuel.Soybean oil may be split, inter-esterified, sulfurized, epoxidized,polymerized, ethoxylated, or cleaved. Designing and producing soybeanoil derivatives with improved functionality and improved oliochemistryis a rapidly growing field. The typical mixture of triglycerides isusually split and separated into pure fatty acids, which are thencombined with petroleum-derived alcohols or acids, nitrogen, sulfonates,chlorine, or with fatty alcohols derived from fats and oils to producethe desired type of oil or fat.

Soybeans are also used as a food source for both animals and humans.Soybeans are widely used as a source of protein for poultry, swine, andcattle feed. During processing of whole soybeans, the fibrous hull isremoved and the oil is extracted. The remaining soybean meal is acombination of carbohydrates and approximately 50% protein.

For human consumption, soybean meal is made into soybean flour, which isprocessed to protein concentrates used for meat extenders or specialtypet foods. Production of edible protein ingredients from soybean offersa healthy, less expensive replacement for animal protein in meats, aswell as dairy type products.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope. In various embodiments, one or more ofthe above-described problems have been reduced or eliminated, whileother embodiments are directed to other improvements.

According to the invention, there is provided a new soybean cultivardesignated 03330181. This invention thus relates to the seeds of soybeancultivar 03330181, to the plants of soybean cultivar 03330181 and tomethods for producing a soybean plant produced by crossing soybeancultivar 03330181 with itself or another soybean cultivar, and thecreation of variants by mutagenesis or transformation of soybeancultivar 03330181.

This invention also relates to methods for introgressing a transgenic ormutant trait into soybean cultivar 03330181 and to the soybean plantsand plant parts produced by those methods. This invention also relatesto soybean cultivars or breeding cultivars and plant parts derived fromsoybean cultivar 03330181, to methods for producing other soybeancultivars or plant parts derived from soybean cultivar 03330181 and tothe soybean plants, varieties, and their parts derived from the use ofthose methods. This invention further relates to soybean seeds, plants,and plant parts produced by crossing soybean cultivar 03330181 withanother soybean cultivar. Thus, any such methods using the soybeancultivar 03330181 are part of this invention: selfing, backcrosses,hybrid production, crosses to populations, and the like. All plantsproduced using soybean cultivar 03330181 as at least one parent arewithin the scope of this invention. Advantageously, the soybean cultivarcould be used in crosses with other, different, soybean plants toproduce first generation (F₁) soybean hybrid seeds and plants withsuperior characteristics.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of soybean plant 03330181. The tissue culture willpreferably be capable of regenerating plants having all themorphological and physiological characteristics of the foregoing soybeanplant, and of regenerating plants having substantially the same genotypeas the foregoing soybean plant. Preferably, the regenerable cells insuch tissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, ovules, anthers, cotyledons, hypocotyl, pistils,roots, root tips, flowers, seeds, petiole, pods, or stems. Stillfurther, the present invention provides soybean plants regenerated fromthe tissue cultures of the invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION OF THE INVENTION

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Abiotic stress: As used herein, abiotic stress relates to all non-livingchemical and physical factors in the environment. Examples of abioticstress include, but are not limited to, drought, flooding, salinity,temperature, and climate change.

Allele. Any of one or more alternative forms of a genetic sequence. In adiploid cell or organism, the two alleles of a given sequence typicallyoccupy corresponding loci on a pair of homologous chromosomes.

Alter. The utilization of up-regulation, down-regulation, or genesilencing.

Backcrossing. A process in which a breeder crosses progeny back to oneof the parental genotypes one or more times. Commonly used to introduceone or more locus conversions from one genetic background into another.

Breeding. The genetic manipulation of living organisms.

BU/A. Bushels per Acre. The seed yield in bushels/acre is the actualyield of the grain at harvest.

Brown Stem Rot. This is a visual disease score from 1 to 9 comparing allgenotypes in a given test. The score is based on leaf symptoms ofyellowing and necrosis caused by brown stem rot. Visual scores rangefrom a score of 9, which indicates no symptoms, to a score of 1 whichindicates severe symptoms of leaf yellowing and necrosis.

Cell. Cell as used herein includes a plant cell, whether isolated, intissue culture, or incorporated in a plant or plant part.

Cotyledon. A cotyledon is a type of seed leaf. The cotyledon containsthe food storage tissues of the seed.

Cross-pollination. Fertilization by the union of two gametes fromdifferent plants.

Diploid. A cell or organism having two sets of chromosomes.

Embryo. The embryo is the small plant contained within a mature seed.

Emergence. This score indicates the ability of the seed to emerge whenplanted 3″ deep in sand at a controlled temperature of 25° C. The numberof plants that emerge each day are counted. Based on this data, eachgenotype is given a 1 to 9 score based on its rate of emergence andpercent of emergence. A score of 9 indicates an excellent rate andpercent of emergence, an intermediate score of 5 indicates averageratings and a score of 1 indicates a very poor rate and percent ofemergence.

F_(#). The “F” symbol denotes the filial generation, and the # is thegeneration number, such as F₁, F₂, F₃, etc.

Gene. As used herein, “gene” refers to a unit of inheritancecorresponding to DNA or RNA that code for a type of protein or for anRNA chain that has a function in the organism.

Gene Silencing. The interruption or suppression of the expression of agene at the level of transcription or translation.

Genotype. Refers to the genetic constitution of a cell or organism.

Haploid. A cell or organism having one set of the two sets ofchromosomes in a diploid.

Hilum. This refers to the scar left on the seed that marks the placewhere the seed was attached to the pod prior to the seed beingharvested.

Hypocotyl. A hypocotyl is the portion of an embryo or seedling betweenthe cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

Iron Deficiency Chlorosis. Iron deficiency chlorosis (IDC) is ayellowing of the leaves caused by a lack of iron in the soybean plant.Iron is essential in the formation of chlorophyll, which gives plantstheir green color. In high pH soils, iron becomes insoluble and cannotbe absorbed by plant roots. Soybean cultivars differ in their geneticability to utilize the available iron. A score of 9 means no stunting ofthe plants or yellowing of the leaves, and a score of 1 indicates theplants are dead or dying caused by iron deficiency, a score of 5 meansplants have intermediate health with some leaf yellowing.

Linkage. Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

Linkage Disequilibrium. Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

Linoleic Acid Percent. Linoleic acid is one of the five most abundantfatty acids in soybean seeds. It is measured by gas chromatography andis reported as a percent of the total oil content.

Locus. A defined segment of DNA.

Lodging Resistance. Lodging is rated on a scale of 1 to 9. A score of 9indicates erect plants. A score of 5 indicates plants are leaning at a45° angle in relation to the ground and a score of 1 indicates plantsare lying on the ground.

Maturity Date. Plants are considered mature when 95% of the pods havereached their mature color. The number of days are calculated eitherfrom August 31 or from the planting date.

Maturity Group. This refers to an agreed upon industry division ofgroups of soybean varieties based on zones in which they are adapted,primarily according to day length or latitude. They consist of very longday length varieties (Groups 000, 00, 0), and extend to very short daylength varieties (Groups VII, VIII, IX, X).

Nucleic Acid. An acidic, chainlike biological macromolecule consistingof multiple repeat units of phosphoric acid, sugar and purine andpyrimidine bases.

Relative Maturity (RM). The term relative maturity is a numerical valuethat is assigned to a soybean cultivar based on comparisons with thematurity values of other varieties. The number preceding the decimalpoint in the RM refers to the maturity group. The number following thedecimal point refers to the relative earliness or lateness within eachmaturity group. For example, a 3.0 is an early group III cultivar, whilea 3.9 is a late group III cultivar.

Oil or Oil Percent. Soybean seeds contain a considerable amount of oil.Oil is measured by NIR spectrophotometry and is reported as a percentagebasis.

Oleic Acid Percent. Oleic acid is one of the five most abundant fattyacids in soybean seeds. It is measured by gas chromatography and isreported as a percent of the total oil content.

Palmitic Acid Percent. Palmitic acid is one of the five most abundantfatty acids in soybean seeds. It is measured by gas chromatography andis reported as a percent of the total oil content.

Pedigree. Refers to the lineage or genealogical descent of a plant.

Pedigree Distance. Relationship among generations based on theirancestral links as evidenced in pedigrees. May be measured by thedistance of the pedigree from a given starting point in the ancestry.

Percent Identity. Percent identity as used herein refers to thecomparison of the homozygous alleles of two soybean varieties. Percentidentity is determined by comparing a statistically significant numberof the homozygous alleles of two developed varieties. For example, apercent identity of 90% between soybean cultivar 1 and soybean cultivar2 means that the two cultivars have the same allele at 90% of theirloci.

Percent Similarity. Percent similarity as used herein refers to thecomparison of the homozygous alleles of a soybean cultivar such assoybean cultivar 03330181 with another plant, and if the homozygousallele of soybean cultivar 03330181 matches at least one of the allelesfrom the other plant, then they are scored as similar. Percentsimilarity is determined by comparing a statistically significant numberof loci and recording the number of loci with similar alleles as apercentage. A percent similarity of 90% between soybean cultivar03330181 and another plant means that soybean cultivar 03330181 matchesat least one of the alleles of the other plant at 90% of the loci.

Phytophthora Tolerance. Tolerance to Phytophthora root rot is rated on ascale of 1 to 9, with a score of 9 being the best or highest toleranceranging down to a score of 1 which indicates the plants have notolerance to Phytophthora.

Phenotypic Score. The Phenotypic Score is a visual rating of generalappearance of the cultivar. All visual traits are considered in thescore including healthiness, standability, appearance, and freedom ofdisease. Ratings are scored from 1 being poor to 9 being excellent.

Plant. As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant from which seed,grain, or anthers have been removed. Seed or embryo that will producethe plant is also considered to be the plant.

Plant Height. Plant height is taken from the top of the soil to the topnode of the plant and is measured in centimeters.

Plant Parts. As used herein, the term “plant parts” (or a soybean plant,or a part thereof) includes but is not limited to protoplasts, leaves,stems, roots, root tips, anthers, pistils, seed, grain, embryo, pollen,ovules, cotyledon, hypocotyl, pod, flower, shoot, tissue, petiole,cells, meristematic cells, and the like.

Pod. This refers to the fruit of a soybean plant. It consists of thehull or shell (pericarp) and the soybean seeds.

Progeny. As used herein, includes an F₁ soybean plant produced from thecross of two soybean plants where at least one plant includes soybeancultivar 03330181 and progeny further includes, but is not limited to,subsequent F₂, F₃, F₄, F₅, F₆, F₇, F₈, F₉, and F₁₀ generational crosseswith the recurrent parental line.

Protein Percent. Soybean seeds contain a considerable amount of protein.Protein is generally measured by NIR spectrophotometry and is reportedon an as is percentage basis.

Pubescence. This refers to a covering of very fine hairs closelyarranged on the leaves, stems, and pods of the soybean plant.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Seed Protein Peroxidase Activity. Seed protein peroxidase activityrefers to a chemical taxonomic technique to separate cultivars based onthe presence or absence of the peroxidase enzyme in the seed coat. Thereare two types of soybean cultivars; those having high peroxidaseactivity (dark red color) and those having low peroxidase activity (nocolor).

Seed Yield (Bushels/Acre). The yield in bushels/acre is the actual yieldof the grain at harvest.

Seeds Per Pound. Soybean seeds vary in seed size; therefore, the numberof seeds required to make up one pound also varies. The number of seedsper pound affect the pounds of seed required to plant a given area andcan also impact end uses.

Shattering. The amount of pod dehiscence prior to harvest. Poddehiscence involves seeds falling from the pods to the soil. This is avisual score from 1 to 9 comparing all genotypes within a given test. Ascore of 9 means pods have not opened and no seeds have fallen out. Ascore of 5 indicates approximately 50% of the pods have opened, withseeds falling to the ground, and a score of 1 indicates 100% of the podsare opened.

Single Locus Converted (Conversion). Single locus converted(conversion), also known as coisogenic plants, refers to plants whichare developed by a plant breeding technique called backcrossing and/orby genetic transformation to introduce a given locus that is transgenicin origin, wherein essentially all of the morphological andphysiological characteristics of a soybean variety are recovered inaddition to the characteristics of the locus transferred into thevariety via the backcrossing technique or by genetic transformation.

Breeding History

The breeding history of the cultivar can be summarized as follows:

-   -   2015-16 A cross with parentage 13515229-01×14535248 was made        near Rancagua, Chile.    -   2016 An F1 population was grown near Adel, Iowa    -   2016-17 An F2 populations were grown near Chacabuco, Argentina.    -   2017 F3 bulk populations were grown in the Midwest and single        plants were pulled.    -   2017-18 Plant rows were grown near Chacabuco, Argentina.    -   2018 Yield trials were grown at 6 locations in the Midwest. F5        single plants were pulled.

2018-19 Plant rows were grown near Chacabuco, Argentina.

-   -   2019 Based on yield from 2018 trials, 16MD20441-81-03 was        advanced to 2019 PRYT yield trials.    -   2020 Based on yield from 2019 trials, 16MD20441-81-03 was        advanced to 2020 Elite yield trials.

16MD20441-81-03 given variety designation 03330181.

The cultivar has shown uniformity and stability, as described in thefollowing variety description information. It has been self-pollinated asufficient number of generations with careful attention to uniformity ofplant type. The line has been increased with continued observation foruniformity. The results of an objective evaluation of the cultivar arepresented in the table(s) that follow.

TABLE 1 DESCRIPTION OF SOYBEAN CULTIVAR 03330181 Seed Coat Color (MatureSeed): Yellow Seed Coat Luster (Mature Seed): Dull Cotyledon Color(Mature Seed): Yellow Leaflet Shape: Ovate Growth Habit: IndeterminateFlower Color: Purple Hilum Color (Mature Seed): Buff Plant PubescenceColor: Gray Pod Wall Color: Tan Maturity Group: II Relative Maturity:2.8 Plant Lodging Score: 7.7 Plant Height (cm): 84 Seed Size (#seed/lb): 3086 Seed % Protein: 34.9 Seed % Oil: 19.7

Physiological Responses: Contains DAS-44406-6 event conferring toleranceto 2,4-D herbicides, glyphosate herbicides, and glufosinate herbicides.Event DAS-44406-6 is the subject of U.S. Pat. Nos. 5,491,288; 5,510,471;5,633,448; 5,717,084; 5,728,925; 5,792,930; 6,063,601; 6,313,282;6,338,961; 6,566,587; 8,283,522; 8,460,891; 8,916,752; 9,371,394; and9,540,655, the disclosures of which are incorporated herein byreference. There is natural variation in soybeans caused by genetics andenvironment. Soybeans containing Event DAS-44406-6 (Enlist E3™ soybeans)are genetically yellow. From time to time, seed coat color variation maybe observed in soybean seeds comprising Event DAS-44406-6 due toenvironment and other factors. This can include a light brown bandconnecting the ends of the hilum and/or light brown shadows on each sideof the hilum of the yellow seed coat. Such seeds are however hereindescribed based on genetics, i.e., as having yellow seed coat (hull) andyellow cotyledons.

Disease Resistance: Soybean Cyst Nematode—rhg 1.

This invention is also directed to methods for producing a soybean plantby crossing a first parent soybean plant with a second parent soybeanplant, wherein the first or second soybean plant is the soybean plantfrom cultivar 03330181. Further, both first and second parent soybeanplants may be from cultivar 03330181. Therefore, any methods usingsoybean cultivar 03330181 are part of this invention: selfing,backcrosses, hybrid breeding, and crosses to populations. Any plantsproduced using soybean cultivar 03330181 as at least one parent arewithin the scope of this invention.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformant plants obtained with the germplasm of the invention areintended to be within the scope of this invention.

Soybean cultivar 03330181 is similar to soybean cultivar 13515229-01.While similar to soybean cultivar 13515229-01 there are numerousdifferences including: soybean cultivar 03330181 has buff hila color,while soybean cultivar 13515229-01 has imperfect black hila color.

Further Embodiments of the Invention

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to alter the traits of a plant in a specific manner.Any DNA sequences, whether from a different species or from the samespecies, which are introduced into the genome using transformation orvarious breeding methods are referred to herein collectively as“transgenes.” In some embodiments of the invention, a transgenic variantof soybean cultivar 03330181 may contain at least one transgene butcould contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and/or no morethan 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. Over the last 15to 20 years several methods for producing transgenic plants have beendeveloped, and the present invention also relates to transgenic variantsof the claimed soybean cultivar 03330181.

Nucleic acids or polynucleotides refer to RNA or DNA that is linear orbranched, single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids. These terms also encompass untranslatedsequence located at both the 3′ and 5′ ends of the coding region of thegene: at least approximately 1000 nucleotides of sequence upstream fromthe 5′ end of the coding region and at least approximately 200nucleotides of sequence downstream from the 3′ end of the coding regionof the gene. Less common bases, such as inosine, 5-methylcytosine,6-methyladenine, hypoxanthine and others can also be used for antisense,dsRNA and ribozyme pairing. For example, polynucleotides that containC-5 propyne analogues of uridine and cytidine have been shown to bindRNA with high affinity and to be potent antisense inhibitors of geneexpression. Other modifications, such as modification to thephosphodiester backbone, or the 2′-hydroxy in the ribose sugar group ofthe RNA can also be made. The antisense polynucleotides and ribozymescan consist entirely of ribonucleotides, or can contain mixedribonucleotides and deoxyribonucleotides. The polynucleotides of theinvention may be produced by any means, including genomic preparations,cDNA preparations, in vitro synthesis, RT-PCR, and in vitro or in vivotranscription.

One embodiment of the invention is a process for producing soybeancultivar 03330181 further comprising a desired trait, said processcomprising introducing a transgene that confers a desired trait to asoybean plant of cultivar 03330181. Another embodiment is the productproduced by this process. In one embodiment the desired trait may be oneor more of herbicide resistance, insect resistance, disease resistance,decreased phytate, or modified fatty acid or carbohydrate metabolism.The specific gene may be any known in the art or listed herein,including: a polynucleotide conferring resistance to imidazolinone,dicamba, sulfonylurea, glyphosate, glufosinate, triazine, benzonitrile,cyclohexanedione, phenoxy propionic acid, and L-phosphinothricin; apolynucleotide encoding a Bacillus thuringiensis polypeptide; apolynucleotide encoding phytase, FAD-2, FAD-3, galactinol synthase, or araffinose synthetic enzyme; or a polynucleotide conferring resistance tosoybean cyst nematode, brown stem rot, Phytophthora root rot, soybeanmosaic virus, or sudden death syndrome.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants,” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993), andArmstrong, “The First Decade of Maize Transformation: A Review andFuture Perspective,” Maydica, 44:101-109 (1999). In addition, expressionvectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation,” in Methodsin Plant Molecular Biology and Biotechnology, Glick and Thompson Eds.,CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A genetic trait which has been engineered into the genome of aparticular soybean plant may then be moved into the genome of anothercultivar using traditional breeding techniques that are well known inthe plant breeding arts. For example, a backcrossing approach iscommonly used to move a transgene from a transformed soybean cultivarinto an already developed soybean cultivar, and the resulting backcrossconversion plant would then comprise the transgene(s).

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to, genes,coding sequences, inducible, constitutive and tissue specific promoters,enhancing sequences, and signal and targeting sequences. For example,see the traits, genes, and transformation methods listed in U.S. Pat.No. 6,118,055.

Included among various plant transformation techniques are methods thatpermit the site-specific modification of a plant genome, includingcoding sequences, regulatory elements, non-coding and other DNAsequences in a plant genome. Such methods are well-known in the art andinclude, for example, use of the CRISPR-Cas system, zinc-fingernucleases (ZFNs), and transcription activator-like effector nucleases(TALENs), among others.

Plant transformation may involve the construction of an expressionvector which will function in plant cells. Such a vector can compriseDNA comprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid and can beused alone or in combination with other plasmids to provide transformedsoybean plants using transformation methods as described below toincorporate transgenes into the genetic material of the soybeanplant(s).

Expression Vectors for Soybean Transformation: Marker Genes

Expression vectors include at least one genetic marker operably linkedto a regulatory element (for example, a promoter) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant (Hayford et al., PlantPhysiol., 86:1216 (1988); Jones et al., Mol. Gen. Genet., 210:86 (1987);Svab et al., Plant Mol. Biol., 14:197 (1990); Hille et al., Plant Mol.Biol., 7:171 (1986)). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate, or bromoxynil (Comai et al.,Nature, 317:741-744 (1985); Gordon-Kamm et al., Plant Cell, 2:603-618(1990); Stalke et al., Science, 242:419-423 (1988)).

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactatesynthase (Eichholtz et al., Somatic Cell Mol. Genet., 13:67 (1987); Shahet al., Science, 233:478 (1986); Charest et al., Plant Cell Rep., 8:643(1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells, rather than directgenetic selection of transformed cells, for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep., 5:387 (1987); Teeri et al.,EMBO J., 8:343 (1989); Koncz et al., Proc. Natl. Acad. Sci. USA, 84:131(1987); DeBlock et al., EMBO J., 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available (Molecular Probes, Publication2908, IMAGENE GREEN, pp. 1-4 (1993); Naleway et al., J. Cell Biol.,115:151a (1991)). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds, andlimitations associated with the use of luciferase genes as selectablemarkers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells (Chalfie et al., Science, 263:802 (1994)). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Soybean Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific.” A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell-type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in soybean. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in soybean. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See, Wardet al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Mett et al., Proc. Natl. Acad. Sci. USA,90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Hershey et al., Mol. GenGenetics, 227:229-237 (1991); Gatz et al., Mol. Gen. Genetics, 243:32-38(1994)); or Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genetics,227:229-237 (1991)). A particularly preferred inducible promoter is apromoter that responds to an inducing agent to which plants do notnormally respond. An exemplary inducible promoter is the induciblepromoter from a steroid hormone gene, glucocorticoid response elements,the transcriptional activity of which is induced by a glucocorticoidhormone (Schena et al., Proc. Natl. Acad. Sci. USA, 88:10421-10425(1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in soybean or the constitutive promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in soybean.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell, 2: 163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol., 12:619-632 (1989);Christensen et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten et al., EMBOJ., 3:2723-2730 (1984)); and maize H3 histone (Lepetit et al., Mol. Gen.Genetics, 231:276-285 (1992); Atanassova et al., Plant Journal, 2 (3):291-300 (1992)). The ALS promoter, an Xba1/Ncol fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xba1/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT Application WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in soybean.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in soybean. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promotersuch as that from the phaseolin gene (Murai et al., Science, 23:476-482(1983); Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA,82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J., 4(11):2723-2729(1985); Timko et al., Nature, 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics,217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics, 244:161-168 (1993)); or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of a protein produced by transgenes to a subcellularcompartment, such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall, or mitochondrion, or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine during protein synthesis andprocessing where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker et al., Plant Mol. Biol., 20:49 (1992); Knox, C. et al.,Plant Mol. Biol., 9:3-17 (1987); Lerner et al., Plant Physiol.,91:124-129 (1989); Frontes et al., Plant Cell, 3:483-496 (1991);Matsuoka et al., Proc. Natl. Acad. Sci., 88:834 (1991); Gould et al., J.Cell. Biol., 108:1657 (1989); Creissen et al., Plant J., 2:129 (1991);Kalderon et al., Cell, 39:499-509 (1984); Steifel et al., Plant Cell,2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein can then beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a soybean plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see, Glick and Thompson, Methods in PlantMolecular Biology and Biotechnology, CRC Press, Inc., Boca Raton,269:284 (1993). Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant.

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome,” Science,280:1077-1082 (1998), and similar capabilities are becoming increasinglyavailable for the soybean genome. Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR, and sequencing, all of which areconventional techniques. SNPs may also be used alone or in combinationwith other techniques.

Likewise, by means of the present invention, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Throughthe transformation of soybean, the expression of genes can be altered toenhance disease resistance, insect resistance, herbicide resistance,agronomic, grain quality, and other traits. Transformation can also beused to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to soybean, as well as non-nativeDNA sequences, can be transformed into soybean and used to alter levelsof native or non-native proteins. Various promoters, targetingsequences, enhancing sequences, and other DNA sequences can be insertedinto the genome for the purpose of altering the expression of proteins.Reduction of the activity of specific genes (also known as genesilencing or gene suppression) is desirable for several aspects ofgenetic engineering in plants.

Many techniques for gene silencing are well known to one of skill in theart, including, but not limited to, knock-outs (such as by insertion ofa transposable element such as mu (Vicki Chandler, The Maize Handbook,Ch. 118 (Springer-Verlag 1994)) or other genetic elements such as a FRTand Lox that are used for site specific integrations, antisensetechnology (see, e.g., Sheehy et al., PNAS USA, 85:8805-8809 (1988); andU.S. Pat. Nos. 5,107,065, 5,453,566, and 5,759,829); co-suppression(e.g., Taylor, Plant Cell, 9:1245 (1997); Jorgensen, Trends Biotech.,8(12):340-344 (1990); Flavell, PNAS USA, 91:3490-3496 (1994); Finneganet al., Bio/Technology, 12:883-888 (1994); Neuhuber et al., Mol. Gen.Genet., 244:230-241 (1994)); RNA interference (Napoli et al., PlantCell, 2:279-289 (1990); U.S. Pat. No. 5,034,323; Sharp, Genes Dev.,13:139-141 (1999); Zamore et al., Cell, 101:25-33 (2000); Montgomery etal., PNAS USA, 95:15502-15507 (1998)), virus-induced gene silencing(Burton et al., Plant Cell, 12:691-705 (2000); Baulcombe, Curr. Op.Plant Bio., 2:109-113 (1999)); target-RNA-specific ribozymes (Haseloffet al., Nature, 334: 585-591 (1988)); hairpin structures (Smith et al.,Nature, 407:319-320 (2000); WO 99/53050; WO 98/53083); MicroRNA(Aukerman & Sakai, Plant Cell, 15:2730-2741 (2003)); ribozymes(Steinecke et al., EMBO J., 11:1525 (1992); Perriman et al., AntisenseRes. Dev., 3:253 (1993)); oligonucleotide mediated targeted modification(e.g., WO 03/076574 and WO 99/25853); Zn-finger targeted molecules(e.g., WO 01/52620, WO 03/048345, and WO 00/42219); and other methods orcombinations of the above methods known to those of skill in the art.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant cultivar can be transformed with one ormore cloned resistance genes to engineer plants that are resistant tospecific pathogen strains. See, for example, Jones et al., Science,266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin et al., Science, 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. tomato encodes aprotein kinase); Mindrinos et al., Cell, 78:1089 (1994) (ArabidopsisRSP2 gene for resistance to Pseudomonas syringae); McDowell & Woffenden,Trends Biotechnol., 21(4):178-83 (2003); and Toyoda et al., TransgenicRes., 11 (6):567-82 (2002).

B. A gene conferring resistance to a pest, such as soybean cystnematode. See, e.g., PCT Application WO 96/30517 and PCT Application WO93/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modelled thereon. See, for example, Geiser et al., Gene,48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995, and 31998.

D. A lectin. See, for example, Van Damme et al., Plant Molec. Biol.,24:25 (1994), who disclose the nucleotide sequences of several Cliviaminiata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See, PCT Application US93/06487, which teaches the use of avidin and avidin homologues aslarvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub et al., Plant Molec. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani etal., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor); and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone, such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor); Pratt et al.,Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata); Chattopadhyay et al., CriticalReviews in Microbiology, 30(1):33-54 (2004); Zjawiony, J Nat Prod,67(2):300-310 (2004); Carlini & Grossi-de-Sa, Toxicon, 40(11):1515-1539(2002); Ussuf et al., Curr Sci., 80(7):847-853 (2001); Vasconcelos &Oliveira, Toxicon, 44(4):385-403 (2004). See also, U.S. Pat. No.5,266,317 to Tomalski et al., which discloses genes encodinginsect-specific, paralytic neurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see, Pang et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See, PCTApplication WO 93/02197 (Scott et al.), which discloses the nucleotidesequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also, Kramer et al., InsectBiochem. Molec. Biol., 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol., 21:673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene, U.S. Pat. Nos. 7,145,060,7,087,810, and 6,563,020.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See, PCT Application WO 95/16776 andU.S. Pat. No. 5,580,852, which disclose peptide derivatives oftachyplesin which inhibit fungal plant pathogens, and PCT Application WO95/18855 and U.S. Pat. No. 5,607,914 which teaches syntheticantimicrobial peptides that confer disease resistance.

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci, 89:43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,and tobacco mosaic virus.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. See,Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See, Lamb et al., Bio/Technology,10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J., 2:367 (1992).

S. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology, 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

T. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis-related genes. Briggs, S., Current Biology, 5(2)(1995); Pieterse & Van Loon, Curr. Opin. Plant Bio., 7(4):456-64 (2004);and Somssich, Cell, 113(7):815-6 (2003).

U. Antifungal genes. See, Cornelissen and Melchers, Plant Physiol.,101:709-712 (1993); Parijs et al., Planta, 183:258-264 (1991); andBushnell et al., Can. J. of Plant Path., 20(2):137-149 (1998). See also,U.S. Pat. No. 6,875,907.

V. Detoxification genes, such as for fumonisin, beauvericin,moniliformin, and zearalenone and their structurally-relatedderivatives. See, for example, U.S. Pat. No. 5,792,931.

W. Cystatin and cysteine proteinase inhibitors. See, U.S. Pat. No.7,205,453.

X. Defensin genes. See, WO 03/000863 and U.S. Pat. No. 6,911,577.

Y. Genes conferring resistance to nematodes, and in particular soybeancyst nematodes. See, e.g., PCT Applications WO 96/30517, WO 93/19181,and WO 03/033651; Urwin et al., Planta, 204:472-479 (1998); Williamson,Curr Opin Plant Bio., 2(4):327-31 (1999).

Z. Genes that confer resistance to Phytophthora Root Rot, such as theRps1, Rps1a, Rps1b, Rps1c, Rps1d, Rps1e, Rps1k, Rps2, Rps3a, Rps3b,Rps3c, Rps4, Rps5, Rps6, Rps7, and other Rps genes. See, for example,Shoemaker et al., Phytophthora Root Rot Resistance Gene Mapping inSoybean, Plant Genome IV Conference, San Diego, Calif. (1995).

AA. Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.

Any of the above-listed disease or pest resistance genes (A-AA) can beintroduced into the claimed soybean cultivar through a cultivar of meansincluding, but not limited to, transformation and crossing.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J., 7:1241 (1988) and Miki et al., Theor. Appl. Genet., 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds, such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), pyridinoxy or phenoxy propionic acids, andcyclohexanediones (ACCase inhibitor-encoding genes). See, for example,U.S. Pat. No. 4,940,835 to Shah et al., which discloses the nucleotidesequence of a form of EPSPS which can confer glyphosate resistance. U.S.Pat. No. 5,627,061 to Barry et al., also describes genes encoding EPSPSenzymes. See also, U.S. Pat. Nos. 6,566,587, 6,338,961, 6,248,876,6,040,497, 5,804,425, 5,633,435, 5,145,783, 4,971,908, 5,312,910,5,188,642, 4,940,835, 5,866,775, 6,225,114, 6,130,366, 5,310,667,4,535,060, 4,769,061, 5,633,448, 5,510,471, RE 36,449, RE 37,287, and5,491,288; and International Publications EP1173580, WO 01/66704,EP1173581, and EP1173582, which are incorporated herein by reference forthis purpose. Glyphosate resistance is also imparted to plants thatexpress a gene that encodes a glyphosate oxido-reductase enzyme, asdescribed more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, whichare incorporated herein by reference for this purpose. In addition,glyphosate resistance can be imparted to plants by the over expressionof genes encoding glyphosate N-acetyltransferase. See, for example, U.S.Pat. No. 7,462,481. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC Accession No. 39256, and the nucleotide sequence ofthe mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European Patent Appl. No. 0 333 033 to Kumada et al. and U.S. Pat. No.4,975,374 to Goodman et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a PAT gene is provided inEuropean Patent Appl. No. 0 242 246 to Leemans et al. DeGreef et al.,Bio/Technology, 7:61 (1989) describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cyclohexanediones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2, and Acc2-S3 genes described byMarshall et al., Theor. Appl. Genet., 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibila et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.,285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See, Hattori et al., Mol. Gen.Genet., 246:419 (1995). Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17 (1994)); genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol., 36:1687 (1995)); and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol., 20:619(1992)).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837,5,767,373, and International Publication WO 01/12825.

Any of the above listed herbicide genes (A-E) can be introduced into theclaimed soybean cultivar through a variety of means including but notlimited to transformation and crossing.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See, Knultzon et al., Proc. Natl. Acad. Sci.USA, 89:2625 (1992).

B. Decreased phytate content: 1) Introduction of a phytase-encoding geneenhances breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see, Van Hartingsveldt et al., Gene,127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) Up-regulation of a gene that reducesphytate content. In maize, this, for example, could be accomplished bycloning and then re-introducing DNA associated with one or more of thealleles, such as the LPA alleles, identified in maize mutantscharacterized by low levels of phytic acid, such as in Raboy et al.,Maydica, 35:383 (1990), and/or by altering inositol kinase activity asin WO 02/059324, U.S. Publ. No. 2003/000901, WO 03/027243, U.S. Publ.No. 2003/0079247, WO 99/05298, U.S. Pat. Nos. 6,197,561, 6,291,224,6,391,348, WO 2002/059324, U.S. Publ. No. 2003/0079247, WO 98/45448, WO99/55882, and WO 01/04147.

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch, or a gene altering thioredoxin, such as NTRand/or TRX (see, U.S. Pat. No. 6,531,648, which is incorporated byreference for this purpose), and/or a gamma zein knock out or mutant,such as cs27 or TUSC27 or en27 (see, U.S. Pat. Nos. 6,858,778 and7,741,533 and U.S. Publ. No. 2005/0160488, which are incorporated byreference for this purpose). See, Shiroza et al., J. Bacteriol., 170:810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene); Steinmetz et al., Mol. Gen. Genet., 200:220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene); Pen et al.,Bio/Technology, 10:292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis alpha-amylase); Elliot et al., PlantMolec. Biol., 21:515 (1993) (nucleotide sequences of tomato invertasegenes); Sogaard et al., J. Biol. Chem., 268:22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene); Fisher et al., PlantPhysiol., 102:1045 (1993) (maize endosperm starch branching enzyme II);WO 99/10498 (improved digestibility and/or starch extraction throughmodification of UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref 1, HCHL,C4H); U.S. Pat. No. 6,232,529 (method of producing high oil seed bymodification of starch levels (AGP)). The fatty acid modification genesmentioned above may also be used to affect starch content and/orcomposition through the interrelationship of the starch and oilpathways.

D. Elevated oleic acid via FAD-2 gene modification and/or decreasedlinolenic acid via FAD-3 gene modification. See, U.S. Pat. Nos.6,063,947, 6,323,392, and International Publication WO 93/11245.Linolenic acid is one of the five most abundant fatty acids in soybeanseeds. The low oxidative stability of linolenic acid is one reason thatsoybean oil undergoes partial hydrogenation. When partiallyhydrogenated, all unsaturated fatty acids form trans fats. Soybeans arethe largest source of edible-oils in the U.S. and 40% of soybean oilproduction is partially hydrogenated. The consumption of trans fatsincreases the risk of heart disease. Regulations banning trans fats haveencouraged the development of low linolenic soybeans. Soybeanscontaining low linolenic acid percentages create a more stable oilrequiring hydrogenation less often. This provides trans-fat freealternatives in products such as cooking oil.

E. Altering conjugated linolenic or linoleic acid content, such as in WO01/12800. Altering LEC1, AGP, Dek1, Superal1, mi1ps, and various Ipagenes, such as Ipa1, Ipa3, hpt, or hggt. See, for example, WO 02/42424,WO 98/22604, WO 03/011015, WO 02/057439, WO 03/011015, U.S. Pat. Nos.6,423,886, 6,197,561, 6,825,397, 7,157,621, U.S. Publ. No. 2003/0079247,and Rivera-Madrid, R. et al., Proc. Natl. Acad. Sci., 92:5620-5624(1995).

F. Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. See, for example, U.S. Pat. Nos. 6,787,683,7,154,029, WO 00/68393 (involving the manipulation of antioxidant levelsthrough alteration of a phytl prenyl transferase (ppt)); WO 03/082899(through alteration of a homogentisate geranyl geranyl transferase(hggt)).

G. Altered essential seed amino acids. See, for example, U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds); U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds); U.S. Pat. No. 5,990,389(high lysine); U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds); U.S. Pat. No. 5,885,802 (high methionine); U.S.Pat. No. 5,885,801 (high threonine); U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes); U.S. Pat. No. 6,459,019 (increasedlysine and threonine); U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit); U.S. Pat. No. 6,346,403 (methionine metabolicenzymes); U.S. Pat. No. 5,939,599 (high sulfur); U.S. Pat. No. 5,912,414(increased methionine); U.S. Pat. No. 5,633,436 (increasing sulfur aminoacid content); U.S. Pat. No. 5,559,223 (synthetic storage proteins withdefined structure containing programmable levels of essential aminoacids for improvement of the nutritional value of plants); U.S. Pat. No.6,194,638 (hemicellulose); U.S. Pat. No. 7,098,381 (UDPGdH); U.S. Pat.No. 6,194,638 (RGP); U.S. Pat. Nos. 6,399,859, 6,930,225, 7,179,955, and6,803,498; U.S. Publ. No. 2004/0068767; WO 99/40209 (alteration of aminoacid compositions in seeds); WO 99/29882 (methods for altering aminoacid content of proteins); WO 98/20133 (proteins with enhanced levels ofessential amino acids); WO 98/56935 (plant amino acid biosyntheticenzymes); WO 98/45458 (engineered seed protein having higher percentageof essential amino acids); WO 98/42831 (increased lysine); WO 96/01905(increased threonine); WO 95/15392 (increased lysine); WO 01/79516; andWO 00/09706 (Ces A: cellulose synthase).

4. Genes that Control Male Sterility:

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describes a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on,”the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See, International Publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See, InternationalPublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See, Paul et al.,Plant Mol. Biol., 19:611-622 (1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341, 6,297,426, 5,478,369,5,824,524, 5,850,014, and 6,265,640, all of which are herebyincorporated by reference.

5. Genes that Create a Site for Site Specific DNA Integration:

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/loxP system.See, for example, Lyznik et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep, 21:925-932 (2003) and WO99/25821, which are hereby incorporated by reference. Other systems thatmay be used include the Gin recombinase of phage Mu (Maeser et al.(1991); Vicki Chandler, The Maize Handbook, Ch. 118 (Springer-Verlag1994)); the Pin recombinase of E. coli (Enomoto et al. (1983)); and theR/RS system of the pSR1 plasmid (Araki et al. (1992)).

6. Genes that Affect Abiotic Stress Resistance:

Genes that affect abiotic stress resistance (including but not limitedto flowering, pod and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see: WO 00/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. Nos. 5,892,009, 5,965,705, 5,929,305,5,891,859, 6,417,428, 6,664,446, 6,706,866, 6,717,034, 6,801,104, WO2000/060089, WO 2001/026459, WO 2001/035725, WO 2001/034726, WO2001/035727, WO 2001/036444, WO 2001/036597, WO 2001/036598, WO2002/015675, WO 2002/017430, WO 2002/077185, WO 2002/079403, WO2003/013227, WO 2003/013228, WO 2003/014327, WO 2004/031349, WO2004/076638, WO 98/09521, and WO 99/38977 describing genes, includingCBF genes and transcription factors effective in mitigating the negativeeffects of freezing, high salinity, and drought on plants, as well asconferring other positive effects on plant phenotype; U.S. Publ. No.2004/0148654 and WO 01/36596, where abscisic acid is altered in plantsresulting in improved plant phenotype, such as increased yield and/orincreased tolerance to abiotic stress; WO 2000/006341, WO 04/090143,U.S. Pat. Nos. 7,531,723 and 6,992,237, where cytokinin expression ismodified resulting in plants with increased stress tolerance, such asdrought tolerance, and/or increased yield. See also, WO 02/02776, WO2003/052063, JP 2002281975, U.S. Pat. No. 6,084,153, WO 01/64898, andU.S. Pat. Nos. 6,177,275 and 6,107,547 (enhancement of nitrogenutilization and altered nitrogen responsiveness). For ethylenealteration, see, U.S. Publ. Nos. 2004/0128719, 2003/0166197, and WO2000/32761. For plant transcription factors or transcriptionalregulators of abiotic stress, see, e.g., U.S. Publ. Nos. 2004/0098764 or2004/0078852.

Other genes and transcription factors that affect plant growth andagronomic traits, such as yield, flowering, plant growth, and/or plantstructure, can be introduced or introgressed into plants. See, e.g., WO97/49811 (LHY), WO 98/56918 (ESD4), WO 97/10339, U.S. Pat. No. 6,573,430(TFL), 6,713,663 (FT), 6,794,560, 6,307,126 (GAI), WO 96/14414 (CON), WO96/38560, WO 01/21822 (VRN1), WO 00/44918 (VRN2), WO 99/49064 (GI), WO00/46358 (FRI), WO 97/29123, WO 99/09174 (D8 and Rht), WO 2004/076638,and WO 004/031349 (transcription factors).

Methods for Soybean Transformation

Numerous methods for plant transformation have been developed includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants,” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in-vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber et al., “Vectors for Plant Transformation,” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science,227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci., 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports, 8:238 (1989). See also, U.S. Pat.No. 5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

B. Direct Gene Transfer—Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation where DNA is carried on the surface of microprojectilesmeasuring 1 to 4 μm. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al., Part. Sci. Technol., 5:27 (1987);Sanford, J. C., Trends Biotech., 6:299 (1988); Klein et al., Bio/Tech.,6:559-563 (1988); Sanford, J. C., Physiol Plant, 7:206 (1990); Klein etal., Biotechnology, 10:268 (1992). See also, U.S. Pat. No. 5,015,580(Christou et al.), issued May 14, 1991 and U.S. Pat. No. 5,322,783(Tomes et al.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology, 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985); Christouet al., Proc Natl. Acad. Sci. USA, 84:3962 (1987). Direct uptake of DNAinto protoplasts using CaCl₂) precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.,199:161 (1985) and Draper et al., Plant Cell Physiol., 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described (Donn et al., In Abstracts of VII^(th) InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990);D'Halluin et al., Plant Cell, 4:1495-1505 (1992); and Spencer et al.,Plant Mol. Biol., 24:51-61 (1994)).

Following transformation of soybean target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues, and/or plants, usingregeneration and selection methods well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic cultivar. The transgenic cultivar could then becrossed with another (non-transformed or transformed) cultivar in orderto produce a new transgenic cultivar. Alternatively, a genetic traitthat has been engineered into a particular soybean line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite cultivar into anelite cultivar, or from a cultivar containing a foreign gene in itsgenome into a cultivar or cultivars that do not contain that gene. Asused herein, “crossing” can refer to a simple x by y cross or theprocess of backcrossing depending on the context.

Genetic Marker Profile Through SSR and First Generation Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same cultivar,or a related cultivar, or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) (which are also referred to asMicrosatellites), and Single Nucleotide Polymorphisms (SNPs). Forexample, see, Cregan et al., “An Integrated Genetic Linkage Map of theSoybean Genome,” Crop Science, 39:1464-1490 (1999) and Berry et al.,“Assessing Probability of Ancestry Using Simple Sequence RepeatProfiles: Applications to Maize Inbred Lines and Soybean Varieties,”Genetics, 165:331-342 (2003), each of which are incorporated byreference herein in their entirety.

Particular markers used for these purposes are not limited to anyparticular set of markers, but are envisioned to include any type ofmarker and marker profile which provides a means of distinguishingvarieties. One method of comparison is to use only homozygous loci forsoybean cultivar 03330181.

Primers and PCR protocols for assaying these and other markers aredisclosed in the Soybase (sponsored by the USDA Agricultural ResearchService and Iowa State University). In addition to being used foridentification of soybean cultivar 03330181, and plant parts and plantcells of soybean cultivar 03330181, the genetic profile may be used toidentify a soybean plant produced through the use of soybean cultivar03330181 or to verify a pedigree for progeny plants produced through theuse of soybean cultivar 03330181. The genetic marker profile is alsouseful in breeding and developing backcross conversions.

The present invention provides in one embodiment a soybean plantcultivar characterized by molecular and physiological data obtained fromthe representative sample of said cultivar deposited with theProvasoli-Guillard National Center for Marine Algae and Microbiota(NCMA). Further provided by the invention is a soybean plant formed bythe combination of the disclosed soybean plant or plant cell withanother soybean plant or cell and comprising the homozygous alleles ofthe cultivar.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, bypolymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. PCR detection is done by use oftwo oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA. Repeated cycles of heat denaturation of the DNA followedby annealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing varieties it is preferable if all SSRprofiles are performed in the same lab.

Primers used are publicly available and may be found in the Soybase orCregan supra. See also, PCT Publication No. WO 99/31964 (NucleotidePolymorphisms in Soybean); U.S. Pat. No. 6,162,967 (Positional Cloningof Soybean Cyst Nematode Resistance Genes); and U.S. Pat. No. 7,288,386(Soybean Sudden Death Syndrome Resistant Soybeans and Methods ofBreeding and Identifying Resistant Plants), the disclosure of which areincorporated herein by reference.

The SSR profile of soybean plant 03330181 can be used to identify plantscomprising soybean cultivar 03330181 as a parent, since such plants willcomprise the same homozygous alleles as soybean cultivar 03330181.Because the soybean cultivar is essentially homozygous at all relevantloci, most loci should have only one type of allele present. Incontrast, a genetic marker profile of an F₁ progeny should be the sum ofthose parents, e.g., if one parent was homozygous for allele x at aparticular locus, and the other parent homozygous for allele y at thatlocus, then the F₁ progeny will be xy (heterozygous) at that locus.Subsequent generations of progeny produced by selection and breeding areexpected to be of genotype x (homozygous), y (homozygous), or xy(heterozygous) for that locus position. When the F₁ plant is selfed orsibbed for successive filial generations, the locus should be either xor y for that position.

In addition, plants and plant parts substantially benefiting from theuse of soybean cultivar 03330181 in their development, such as soybeancultivar 03330181 comprising a backcross conversion, transgene, orgenetic sterility factor, may be identified by having a molecular markerprofile with a high percent identity to soybean cultivar 03330181. Sucha percent identity might be 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to soybean cultivar 03330181.

The SSR profile of soybean cultivar 03330181 can also be used toidentify essentially derived varieties and other progeny varietiesdeveloped from the use of soybean cultivar 03330181, as well as cellsand other plant parts thereof. Such plants may be developed using themarkers identified in WO 00/31964, U.S. Pat. Nos. 6,162,967, and7,288,386. Progeny plants and plant parts produced using soybeancultivar 03330181 may be identified by having a molecular marker profileof at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% geneticcontribution from soybean cultivar, as measured by either percentidentity or percent similarity. Such progeny may be furthercharacterized as being within a pedigree distance of soybean cultivar03330181, such as within 1, 2, 3, 4, or 5 or less cross-pollinations toa soybean plant other than soybean cultivar 03330181 or a plant that hassoybean cultivar 03330181 as a progenitor. Unique molecular profiles maybe identified with other molecular tools such as SNPs and RFLPs.

While determining the SSR genetic marker profile of the plants describedsupra, several unique SSR profiles may also be identified which did notappear in either parent of such plant. Such unique SSR profiles mayarise during the breeding process from recombination or mutation. Acombination of several unique alleles provides a means of identifying aplant cultivar, an F₁ progeny produced from such cultivar, and progenyproduced from such cultivar.

Single-Gene Conversions

When the term “soybean plant” is used in the context of the presentinvention, this also includes any single gene conversions of thatcultivar. The term single gene converted plant as used herein refers tothose soybean plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the morphological andphysiological characteristics of a cultivar are recovered in addition tothe single gene transferred into the cultivar via the backcrossingtechnique. By “essentially all” as used herein in the context ofmorphological and physiological characteristics it is meant that thecharacteristics of a plant are recovered that are otherwise present whencompared in the same environment, other than occasional variant traitsthat might arise during backcrossing or direct introduction of atransgene. It is understood that a locus introduced by backcrossing mayor may not be transgenic in origin, and thus the term backcrossingspecifically includes backcrossing to introduce loci that were createdby genetic transformation.

Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the cultivar. The term “backcrossing”as used herein refers to the repeated crossing of a hybrid progeny backto the recurrent parent, i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8, ormore times to the recurrent parent. The parental soybean plant thatcontributes the gene for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental soybean plant to which the geneor genes from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman & Sleper (1994); Fehr, Principles of CultivarDevelopment, pp. 261-286 (1987)). In a typical backcross protocol, theoriginal cultivar of interest (recurrent parent) is crossed to a secondcultivar (nonrecurrent parent) that carries the single gene of interestto be transferred. The resulting progeny from this cross are thencrossed again to the recurrent parent and the process is repeated untila soybean plant is obtained wherein essentially all of the morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalcultivar. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the genetic, andtherefore the morphological and physiological constitution of theoriginal cultivar. The choice of the particular nonrecurrent parent willdepend on the purpose of the backcross; one of the major purposes is toadd some agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new cultivar but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic. Examples of these traits include, but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185, 5,973,234, and 5,977,445, the disclosures of which arespecifically hereby incorporated by reference.

Introduction of a New Trait or Locus into Soybean Cultivar 03330181

Cultivar 03330181 represents a new base genetic cultivar into which anew locus or trait may be introgressed. Direct transformation andbackcrossing represent two important methods that can be used toaccomplish such an introgression. The term backcross conversion andsingle locus conversion are used interchangeably to designate theproduct of a backcrossing program.

Backcross Conversions of Soybean Cultivar 03330181

A backcross conversion of soybean cultivar 03330181 occurs when DNAsequences are introduced through backcrossing (Hallauer et al., “CornBreeding,” Corn and Corn Improvements, No. 18, pp. 463-481 (1988)), withsoybean cultivar 03330181 utilized as the recurrent parent. Bothnaturally occurring and transgenic DNA sequences may be introducedthrough backcrossing techniques. A backcross conversion may produce aplant with a trait or locus conversion in at least two or morebackcrosses, including at least 2 crosses, at least 3 crosses, at least4 crosses, at least 5 crosses, and the like. Molecular marker assistedbreeding or selection may be utilized to reduce the number ofbackcrosses necessary to achieve the backcross conversion. For example,see, Openshaw, S. J. et al., Marker-assisted Selection in BackcrossBreeding, Proceedings Symposium of the Analysis of Molecular Data, CropScience Society of America, Corvallis, Oreg. (August 1994), where it isdemonstrated that a backcross conversion can be made in as few as twobackcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes ascompared to unlinked genes), the level of expression of the trait, thetype of inheritance (cytoplasmic or nuclear), and the types of parentsincluded in the cross. It is understood by those of ordinary skill inthe art that for single gene traits that are relatively easy toclassify, the backcross method is effective and relatively easy tomanage. (See, Hallauer et al., Corn and Corn Improvement, Sprague andDudley, Third Ed. (1998)). Desired traits that may be transferredthrough backcross conversion include, but are not limited to, sterility(nuclear and cytoplasmic), fertility restoration, nutritionalenhancements, drought tolerance, nitrogen utilization, altered fattyacid profile, low phytate, industrial enhancements, disease resistance(bacterial, fungal, or viral), insect resistance, and herbicideresistance. In addition, an introgression site itself, such as an FRTsite, Lox site, or other site specific integration site, may be insertedby backcrossing and utilized for direct insertion of one or more genesof interest into a specific plant cultivar. A single locus may containseveral transgenes, such as a transgene for disease resistance that, inthe same expression vector, also contains a transgene for herbicideresistance. The gene for herbicide resistance may be used as aselectable marker and/or as a phenotypic trait. A single locusconversion of site specific integration system allows for theintegration of multiple genes at the converted loci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. The backcross is a form ofinbreeding, and the features of the recurrent parent are automaticallyrecovered after successive backcrosses. Poehlman, Breeding Field Crops,p. 204 (1987). Poehlman suggests from one to four or more backcrosses,but as noted above, the number of backcrosses necessary can be reducedwith the use of molecular markers. Other factors, such as a geneticallysimilar donor parent, may also reduce the number of backcrossesnecessary. As noted by Poehlman, backcrossing is easiest for simplyinherited, dominant, and easily recognized traits.

One process for adding or modifying a trait or locus in soybean cultivar03330181 comprises crossing soybean cultivar 03330181 plants grown fromsoybean cultivar 03330181 seed with plants of another soybean cultivarthat comprise the desired trait or locus, selecting F₁ progeny plantsthat comprise the desired trait or locus to produce selected F₁ progenyplants, crossing the selected progeny plants with the soybean cultivar03330181 plants to produce backcross progeny plants, selecting forbackcross progeny plants that have the desired trait or locus and themorphological characteristics of soybean cultivar 03330181 to produceselected backcross progeny plants, and backcrossing to soybean cultivar03330181 three or more times in succession to produce selected fourth orhigher backcross progeny plants that comprise said trait or locus. Themodified soybean cultivar 03330181 may be further characterized ashaving the morphological and physiological characteristics of soybeancultivar 03330181 listed in Table 1 as determined at the 5% significancelevel when grown in the same environmental conditions and/or may becharacterized by percent similarity or identity to soybean cultivar03330181 as determined by SSR markers. The above method may be utilizedwith fewer backcrosses in appropriate situations, such as when the donorparent is highly related or markers are used in the election step.Desired traits that may be used include those nucleic acids known in theart, some of which are listed herein, that will affect traits throughnucleic acid expression or inhibition. Desired loci include theintrogression of FRT, Lox, and other sites for site specificintegration, which may also affect a desired trait if a functionalnucleic acid is inserted at the integration site.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny soybean seed byadding a step at the end of the process that comprises crossing soybeancultivar 03330181 with the introgressed trait or locus with a differentsoybean plant and harvesting the resultant first generation progenysoybean seed.

Tissue Culture

Further reproduction of the cultivar can occur by tissue culture andregeneration. Tissue culture of various tissues of soybeans andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Komatsuda, T. et al., Crop Sci.,31:333-337 (1991); Stephens, P. A. et al., Theor. Appl. Genet.,82:633-635 (1991); Komatsuda, T. et al., Plant Cell, Tissue and OrganCulture, 28:103-113 (1992); Dhir, S. et al., Plant Cell Reports,11:285-289 (1992); Pandey, P. et al., Japan J. Breed., 42:1-5 (1992);and Shetty, K. et al., Plant Science, 81:245-251 (1992); as well as U.S.Pat. No. 5,024,944, issued Jun. 18, 1991 to Collins et al. and U.S. Pat.No. 5,008,200, issued Apr. 16, 1991 to Ranch et al. Thus, another aspectof this invention is to provide cells which upon growth anddifferentiation produce soybean plants having the morphological andphysiological characteristics of soybean cultivar 03330181.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods,petioles, leaves, stems, roots, root tips, anthers, pistils, and thelike. Means for preparing and maintaining plant tissue culture are wellknown in the art. By way of example, a tissue culture comprising organshas been used to produce regenerated plants. U.S. Pat. Nos. 5,959,185,5,973,234, and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

Using Soybean Cultivar 03330181 to Develop Other Soybean Varieties

Soybean varieties such as soybean cultivar 03330181 are typicallydeveloped for use in seed and grain production. However, soybeanvarieties such as soybean cultivar 03330181 also provide a source ofbreeding material that may be used to develop new soybean varieties.Plant breeding techniques known in the art and used in a soybean plantbreeding program include, but are not limited to, recurrent selection,bulk selection, mass selection, backcrossing, pedigree breeding, openpollination breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection, making double haploids,and transformation. Often combinations of these techniques are used. Thedevelopment of soybean varieties in a plant breeding program requires,in general, the development and evaluation of homozygous varieties.There are many analytical methods available to evaluate a new cultivar.The oldest and most traditional method of analysis is the observation ofphenotypic traits, but genotypic analysis may also be used.

Additional Breeding Methods

This invention is directed to methods for producing a soybean plant bycrossing a first parent soybean plant with a second parent soybean plantwherein either the first or second parent soybean plant is cultivar03330181. The other parent may be any other soybean plant, such as asoybean plant that is part of a synthetic or natural population. Anysuch methods using soybean cultivar 03330181 are part of this invention:selfing, sibbing, backcrosses, mass selection, pedigree breeding, bulkselection, hybrid production, crosses to populations, and the like.These methods are well known in the art and some of the more commonlyused breeding methods are described below. Descriptions of breedingmethods can be found in one of several reference books (e.g., Allard,Principles of Plant Breeding (1960); Simmonds, Principles of CropImprovement (1979); Sneep et al. (1979); Fehr, “Breeding Methods forCultivar Development,” Chapter 7, Soybean Improvement, Production andUses, 2nd ed., Wilcox editor (1987)).

The following describes breeding methods that may be used with soybeancultivar 03330181 in the development of further soybean plants. One suchembodiment is a method for developing a cultivar 03330181 progenysoybean plant in a soybean plant breeding program comprising: obtainingthe soybean plant, or a part thereof, of cultivar 03330181, utilizingsaid plant, or plant part, as a source of breeding material, andselecting a soybean cultivar 03330181 progeny plant with molecularmarkers in common with cultivar 03330181 and/or with morphologicaland/or physiological characteristics selected from the characteristicslisted in Tables 1 or 2. Breeding steps that may be used in the soybeanplant breeding program include pedigree breeding, backcrossing, mutationbreeding, and recurrent selection. In conjunction with these steps,techniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example, SSR markers), and the making of double haploidsmay be utilized.

Another method involves producing a population of soybean cultivar03330181 progeny soybean plants, comprising crossing cultivar 03330181with another soybean plant, thereby producing a population of soybeanplants which, on average, derive 50% of their alleles from soybeancultivar 03330181. A plant of this population may be selected andrepeatedly selfed or sibbed with a soybean cultivar resulting from thesesuccessive filial generations. One embodiment of this invention is thesoybean cultivar produced by this method and that has obtained at least50% of its alleles from soybean cultivar 03330181.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see, Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus the invention includes soybeancultivar 03330181 progeny soybean plants comprising a combination of atleast two cultivar 03330181 traits selected from the group consisting ofthose listed in Tables 1 and 2 or the cultivar 03330181 combination oftraits listed in the Summary of the Invention, so that said progenysoybean plant is not significantly different for said traits thansoybean cultivar 03330181 as determined at the 5% significance levelwhen grown in the same environmental conditions. Using techniquesdescribed herein, molecular markers may be used to identify said progenyplant as a soybean cultivar 03330181 progeny plant. Mean trait valuesmay be used to determine whether trait differences are significant, andpreferably the traits are measured on plants grown under the sameenvironmental conditions. Once such a cultivar is developed, its valueis substantial since it is important to advance the germplasm base as awhole in order to maintain or improve traits such as yield, diseaseresistance, pest resistance, and plant performance in extremeenvironmental conditions.

Progeny of soybean cultivar 03330181 may also be characterized throughtheir filial relationship with soybean cultivar 03330181, as forexample, being within a certain number of breeding crosses of soybeancultivar 03330181. A breeding cross is a cross made to introduce newgenetics into the progeny, and is distinguished from a cross, such as aself or a sib cross, made to select among existing genetic alleles. Thelower the number of breeding crosses in the pedigree, the closer therelationship between soybean cultivar 03330181 and its progeny. Forexample, progeny produced by the methods described herein may be within1, 2, 3, 4, or 5 breeding crosses of soybean cultivar 03330181.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which soybean plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,pods, leaves, roots, root tips, anthers, cotyledons, hypocotyls,meristematic cells, stems, pistils, petiole, and the like.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such assoybean cultivar 03330181 and another soybean cultivar having one ormore desirable characteristics that is lacking or which complementssoybean cultivar 03330181. If the two original parents do not provideall the desired characteristics, other sources can be included in thebreeding population. In the pedigree method, superior plants are selfedand selected in successive filial generations. In the succeeding filialgenerations, the heterozygous condition gives way to homogeneousvarieties as a result of self-pollination and selection. Typically inthe pedigree method of breeding, five or more successive filialgenerations of selfing and selection is practiced: F₁ to F₂; F₂ to F₃;F₃ to F₄; F₄ to F₅; etc. After a sufficient amount of inbreeding,successive filial generations will serve to increase seed of thedeveloped cultivar. Preferably, the developed cultivar compriseshomozygous alleles at about 95% or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one cultivar, the donor parent, to adeveloped cultivar called the recurrent parent, which has overall goodagronomic characteristics yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent, but at the same time retain manycomponents of the nonrecurrent parent by stopping the backcrossing at anearly stage and proceeding with selfing and selection. For example, asoybean cultivar may be crossed with another cultivar to produce a firstgeneration progeny plant. The first generation progeny plant may then bebackcrossed to one of its parent varieties to create a BC₁ or BC₂.Progeny are selfed and selected so that the newly developed cultivar hasmany of the attributes of the recurrent parent and yet several of thedesired attributes of the nonrecurrent parent. This approach leveragesthe value and strengths of the recurrent parent for use in new soybeanvarieties.

Therefore, an embodiment of this invention is a method of making abackcross conversion of soybean cultivar 03330181, comprising the stepsof crossing a plant of soybean cultivar 03330181 with a donor plantcomprising a desired trait, selecting an F₁ progeny plant comprising thedesired trait, and backcrossing the selected F₁ progeny plant to a plantof soybean cultivar 03330181. This method may further comprise the stepof obtaining a molecular marker profile of soybean cultivar 03330181 andusing the molecular marker profile to select for a progeny plant withthe desired trait and the molecular marker profile of soybean cultivar03330181. In one embodiment, the desired trait is a mutant gene ortransgene present in the donor parent.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. Soybean cultivar 03330181 is suitablefor use in a recurrent selection program. The method entails individualplants cross pollinating with each other to form progeny. The progenyare grown and the superior progeny selected by any number of selectionmethods, which include individual plant, half-sib progeny, full-sibprogeny, and selfed progeny. The selected progeny are cross pollinatedwith each other to form progeny for another population. This populationis planted and again superior plants are selected to cross pollinatewith each other. Recurrent selection is a cyclical process and thereforecan be repeated as many times as desired. The objective of recurrentselection is to improve the traits of a population. The improvedpopulation can then be used as a source of breeding material to obtainnew varieties for commercial or breeding use, including the productionof a synthetic cultivar. A synthetic cultivar is the resultant progenyformed by the intercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection, seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk, andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self-pollination, directed pollinationcould be used as part of the breeding program.

Mutation Breeding

Mutation breeding is another method of introducing new traits intosoybean cultivar 03330181. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.,cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil)), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in Fehr,“Principles of Cultivar Development,” Macmillan Publishing Company(1993). In addition, mutations created in other soybean plants may beused to produce a backcross conversion of soybean cultivar 03330181 thatcomprises such mutation.

Breeding with Molecular Markers

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs), and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing soybean cultivar 03330181.

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, Molecular Linkage Map ofSoybean (Glycine max L. Merr.), pp. 6.131-6.138 (1993). In S. J. O'Brien(ed.), Genetic Maps: Locus Maps of Complex Genomes, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., developed a moleculargenetic linkage map that consisted of 25 linkage groups with about 365RFLP, 11 RAPD, 3 classical markers, and 4 isozyme loci. See also,Shoemaker, R. C., 1994 RFLP Map of Soybean, pp. 299-309; In R. L.Phillips and I. K. Vasil (ed.), DNA-based markers in plants, KluwerAcademic Press Dordrecht, the Netherlands.

SSR technology is currently the most efficient and practical markertechnology. More marker loci can be routinely used, and more alleles permarker locus can be found, using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatelliteloci in soybean with as many as 26 alleles. (Diwan, N. and Cregan. P.B., Automated sizing of fluorescent-labelled simple sequence repeat(SSR) markers to assay genetic variation in Soybean, Theor. Appl.Genet., 95:220-225 (1997). Single Nucleotide Polymorphisms may also beused to identify the unique genetic composition of the invention andprogeny varieties retaining that unique genetic composition. Variousmolecular marker techniques may be used in combination to enhanceoverall resolution.

Soybean DNA molecular marker linkage maps have been rapidly constructedand widely implemented in genetic studies. One such study is describedin Cregan et al., “An Integrated Genetic Linkage Map of the SoybeanGenome,” Crop Science, 39:1464-1490 (1999). Sequences and PCR conditionsof SSR Loci in Soybean, as well as the most current genetic map, may befound in Soybase on the World Wide Web.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Production of Double Haploids

The production of double haploids can also be used for the developmentof plants with a homozygous phenotype in the breeding program. Forexample, a soybean plant for which soybean cultivar 03330181 is a parentcan be used to produce double haploid plants. Double haploids areproduced by the doubling of a set of chromosomes (1 N) from aheterozygous plant to produce a completely homozygous individual. Forexample, see, Wan et al., “Efficient Production of Doubled HaploidPlants Through Colchicine Treatment of Anther-Derived Maize Callus,”Theoretical and Applied Genetics, 77:889-892 (1989) and U.S. Pat. No.7,135,615. This can be advantageous because the process omits thegenerations of selfing needed to obtain a homozygous plant from aheterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selected line(as female) with an inducer line. Such inducer lines for maize includeStock 6 (Coe, Am. Nat., 93:381-382 (1959); Sharkar and Coe, Genetics,54:453-464 (1966); KEMS (Deimling, Roeber, and Geiger, Vortr.Pflanzenzuchtg, 38:203-224 (1997); or KMS and ZMS (Chalyk, Bylich &Chebotar, MNL, 68:47 (1994); Chalyk & Chebotar, Plant Breeding,119:363-364 (2000)); and indeterminate gametophyte (ig) mutation(Kermicle, Science, 166:1422-1424 (1969). The disclosures of which areincorporated herein by reference.

Methods for obtaining haploid plants are also disclosed in Kobayashi, M.et al., Journ. of Heredity, 71(1):9-14 (1980); Pollacsek, M., Agronomie(Paris) 12(3):247-251 (1992); Cho-Un-Haing et al., Journ. of PlantBiol., 39(3):185-188 (1996); Verdoodt, L. et al., 96(2):294-300(February 1998); Genetic Manipulation in Plant Breeding, ProceedingsInternational Symposium Organized by EUCARPIA, Berlin, Germany (Sep.8-13, 1985); Chalyk et al., Maize Genet Coop., Newsletter 68:47 (1994).

Thus, an embodiment of this invention is a process for making asubstantially homozygous soybean cultivar 03330181 progeny plant byproducing or obtaining a seed from the cross of soybean cultivar03330181 and another soybean plant and applying double haploid methodsto the F₁ seed or F₁ plant or to any successive filial generation. Basedon studies in maize and currently being conducted in soybean, suchmethods would decrease the number of generations required to produce acultivar with similar genetics or characteristics to soybean cultivar03330181. See, Bernardo, R. and Kahler, A. L., Theor. Appl. Genet.,102:986-992 (2001).

In particular, a process of making seed retaining the molecular markerprofile of soybean cultivar 03330181 is contemplated, such processcomprising obtaining or producing F₁ seed for which soybean cultivar03330181 is a parent, inducing doubled haploids to create progenywithout the occurrence of meiotic segregation, obtaining the molecularmarker profile of soybean cultivar 03330181, and selecting progeny thatretain the molecular marker profile of soybean cultivar 03330181.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard (1960); Simmonds (1979); Sneep et al. (1979); Fehr(1987)).

Industrial Uses

The seed of soybean cultivar 03330181, the plant produced from the seed,the hybrid soybean plant produced from the crossing of the cultivar withany other soybean plant, hybrid seed, and various parts of the hybridsoybean plant can be utilized for human food, livestock feed, and as araw material in industry. The soybean seeds produced by soybean cultivar03330181 can be crushed, or a component of the soybean seeds can beextracted, in order to comprise a commodity plant product, such asprotein concentrate, protein isolate, soybean hulls, meal, flour, or oilfor a food or feed product.

Soybean cultivar 03330181 can be used to produce soybean oil. To producesoybean oil, the soybeans harvested from soybean cultivar 03330181 arecracked, adjusted for moisture content, rolled into flakes and the oilis solvent-extracted from the flakes with commercial hexane. The oil isthen refined, blended for different applications, and sometimeshydrogenated. Soybean oils, both liquid and partially hydrogenated, areused domestically and exported, sold as “vegetable oil” or are used in awide variety of processed foods.

Soybean cultivar 03330181 can be used to produce meal. After oil isextracted from whole soybeans harvested from soybean cultivar 03330181,the remaining material or “meal” is “toasted” (a misnomer because theheat treatment is with moist steam) and ground in a hammer mill. Soybeanmeal is an essential element of the American production method ofgrowing farm animals, such as poultry and swine, on an industrial scalethat began in the 1930s; and more recently the aquaculture of catfish.Ninety-eight percent of the U.S. soybean crop is used for livestockfeed. Soybean meal is also used in lower end dog foods. Soybean mealproduced from soybean cultivar 03330181 can also be used to producesoybean protein concentrate and soybean protein isolate.

In addition to soybean meal, soybean cultivar 03330181 can be used toproduce soy flour. Soy flour refers to defatted soybeans where specialcare was taken during desolventizing (not toasted) to minimizedenaturation of the protein and to retain a high Nitrogen SolubilityIndex (NSI) in making the flour. Soy flour is the starting material forproduction of soy concentrate and soy protein isolate. Defatted soyflour is obtained from solvent extracted flakes, and contains less than1% oil. Full-fat soy flour is made from unextracted, dehulled beans, andcontains about 18% to 20% oil. Due to its high oil content, aspecialized Alpine Fine Impact Mill must be used for grinding ratherthan the more common hammer mill. Low-fat soy flour is made by addingback some oil to defatted soy flour. The lipid content varies accordingto specifications, usually between 4.5% and 9%. High-fat soy flour canalso be produced by adding back soybean oil to defatted flour at thelevel of 15%. Lecithinated soy flour is made by adding soybean lecithinto defatted, low-fat or high-fat soy flours to increase theirdispersibility and impart emulsifying properties. The lecithin contentvaries up to 15%.

For human consumption, soybean cultivar 03330181 can be used to produceedible protein ingredients which offer a healthier, less expensivereplacement for animal protein in meats, as well as in dairy-typeproducts. The soybeans produced by soybean cultivar 03330181 can beprocessed to produce a texture and appearance similar to many otherfoods. For example, soybeans are the primary ingredient in many dairyproduct substitutes (e.g., soy milk, margarine, soy ice cream, soyyogurt, soy cheese, and soy cream cheese) and meat substitutes (e.g.,veggie burgers). These substitutes are readily available in mostsupermarkets. Although soy milk does not naturally contain significantamounts of digestible calcium (the high calcium content of soybeans isbound to the insoluble constituents and remains in the soy pulp), manymanufacturers of soy milk sell calcium-enriched products as well. Soy isalso used in tempeh: the beans (sometimes mixed with grain) arefermented into a solid cake.

Additionally, soybean cultivar 03330181 can be used to produce varioustypes of “fillers” in meat and poultry products. Food service, retail,and institutional (primarily school lunch and correctional) facilitiesregularly use such “extended” products, that is, products which containsoy fillers. Extension may result in diminished flavor, but fat andcholesterol are reduced by adding soy fillers to certain products.Vitamin and mineral fortification can be used to make soy productsnutritionally equivalent to animal protein; the protein quality isalready roughly equivalent.

Table 2 compares performance characteristics of soybean cultivar03330181 to selected varieties of commercial value. Shown are thecomparison numbers, cultivar names, performance characteristics, tvalues, and critical t values at the 0.05% and 0.01% levels ofsignificance, respectively.

TABLE 2 PAIRED COMPARISONS Comp # Year # of Loc. # of Obs. Genotype MeanYld t Value Critical t @ .05 Critical t @ .01 1 2020 38 76 03330181 53.52.04* 1.67 2.38 ME2733 51.9 2 2020 38 76 03330181 53.5 1.89* 1.67 2.38PE250X 52.1 3 2020 38 76 03330181 53.5 2.49** 1.67 2.38 PE2670 51.3 42020 38 76 03330181 53.5 2.55** 1.67 2.38 PE2600 51.7 5 2020 38 7603330181 53.5 4.08** 1.67 2.38 PE2519 49.6 6 2020 38 76 03330181 53.52.40** 1.67 2.38 PE280X 51.6 7 2020 38 76 03330181 53.5 2.40** 1.67 2.38PE2511 51.8 8 2020 38 76 03330181 53.5 2.46** 1.67 2.38 PE2610 51.3*Significant at 0.05 level of probability **Significant at 0.01 level ofprobability

As shown in Table 2, soybean cultivar 03330181 yields better than eightcommercial varieties with the increase over most comparisons beingsignificant at the 0.01 level of probability except for the increaseover ME2733 and PE250X being significant at the 0.05 level ofprobability.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

DEPOSIT INFORMATION

Applicant has made a deposit of at least 625 seeds of the claimedsoybean cultivar 03330181 with the Provasoli-Guillard National Centerfor Marine Algae and Microbiota (NCMA), 60 Bigelow Drive, East Boothbay,Me., 04544 USA. The seeds are deposited under NCMA Accession No. ______.The date of the deposit is ______. The deposit has been accepted underthe Budapest Treaty and will be maintained in the NCMA depository for aperiod of 30 years, or 5 years after the most recent request, or for theenforceable life of the patent, whichever is longer, and will bereplaced if necessary during that period. Upon issuance, allrestrictions on the availability to the public of the deposit will beirrevocably removed consistent with all of the requirements of theBudapest Treaty and 37 C.F.R. §§ 1.801-1.809. Applicant does not waiveany infringement of rights granted under this patent or under the PlantVariety Protection Act (7 USC 2321 et seq.).

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

What is claimed:
 1. A plant of soybean cultivar 03330181, representativeseed of said soybean cultivar having been deposited under NCMA AccessionNo. ______.
 2. A seed that produces the plant of claim
 1. 3. A cell ofthe plant of claim
 1. 4. A tissue culture of regenerable cellscomprising the cell of claim
 3. 5. A method of soybean breeding, saidmethod comprising crossing the plant of claim 1 with itself or a secondsoybean plant to produce soybean seed.
 6. The method of claim 5, furtherdefined as comprising crossing a plant of soybean cultivar 03330181 witha second soybean plant of a different genotype to produce hybrid soybeanseed.
 7. An F₁ hybrid soybean seed produced by the method of claim
 6. 8.A method of producing a plant comprising an added desired trait, saidmethod comprising introducing a transgene conferring the desired traitinto the plant of claim
 1. 9. The method of claim 8, wherein the desiredtrait is selected from the group consisting of male sterility, herbicidetolerance, insect or pest resistance, disease resistance, modified fattyacid metabolism, and modified carbohydrate metabolism.
 10. A plantproduced by the method of claim 8 or a selfed progeny thereof, whereinsaid plant or the selfed progeny thereof comprises the transgene andotherwise comprises all of the morphological and physiologicalcharacteristics of soybean cultivar
 03330181. 11. A method ofintroducing a single locus conversion into a soybean plant, said methodcomprising: (a) crossing a plant of soybean cultivar 03330181 with asecond plant comprising a desired single locus to produce F₁ progenyplants, representative seed of said soybean cultivar 03330181 havingbeen deposited under NCMA Accession No. ______; (b) selecting at least afirst progeny plant from step (a) that comprises the single locus toproduce a selected progeny plant; (c) crossing the selected progenyplant from step (b) with a plant of soybean cultivar 03330181 to produceat least a first backcross progeny plant that comprises the singlelocus; and (d) repeating steps (b) and (c) with the first backcrossprogeny plant produced from step (c) used in place of the first progenyplant of step (b) during said repeating, wherein steps (b) and (c) arerepeated until at least a backcross progeny plant is produced comprisingthe single locus conversion.
 12. The method of claim 11, wherein thesingle locus confers a trait selected from the group consisting of malesterility, herbicide tolerance, insect or pest resistance, diseaseresistance, modified fatty acid metabolism, abiotic stress resistance,and modified carbohydrate metabolism.
 13. A soybean plant of soybeancultivar 03330181, further comprising a single locus conversion, whereinthe plant comprises the single locus conversion and otherwise comprisesall of the morphological and physiological characteristics of soybeancultivar 03330181, representative seed of soybean cultivar 03330181having been deposited under NCMA Accession No. ______.
 14. The method ofclaim 6, wherein the method further comprises: (a) crossing a plantgrown from said hybrid soybean seed with itself or a different soybeanplant to produce a seed of a progeny plant of a subsequent generation.15. The method of claim 14, wherein the method further comprises: (b)growing a progeny plant of a subsequent generation from said seed of theprogeny plant of the subsequent generation and crossing the progenyplant of the subsequent generation with itself or a second plant toproduce a progeny plant of a further subsequent generation.
 16. Themethod of claim 15, wherein the method further comprises: (c) repeatingsteps (a) and (b) using said progeny plant of the further subsequentgeneration from step (b) in place of the plant grown from said hybridsoybean seed in step (a), wherein steps (a) and (b) are repeated withsufficient inbreeding to produce an inbred soybean plant derived fromthe soybean cultivar
 03330181. 17. A method of introducing a mutationinto the genome of soybean cultivar 03330181, said method comprisingapplying a mutagen to the plant of claim 1, or a part thereof, whereinsaid mutagen is selected from the group consisting of ethylmethanesulfonate, gamma-rays, and sodium azide, and wherein theresulting plant comprises a genome mutation.
 18. A mutagenized soybeanplant produced by the method of claim 17, wherein the mutagenizedsoybean plant comprises a mutation in the genome and otherwise comprisesall of the morphological and physiological characteristics of soybeancultivar
 03330181. 19. A method of producing a commodity plant product,said method comprising obtaining the plant of claim 1 or a part thereofand producing said commodity plant product therefrom.
 20. The method ofclaim 19, wherein the commodity plant product is protein concentrate,protein isolate, soybean hulls, meal, flour, or oil.